Transferrin is a well known iron-binding protein, which is responsible for carrying iron into the cells via its binding to the transferrin receptor. Moreover, transferrin has been widely used as carrier for anti-cancer drug delivery systems since the transferrin-receptor is upregulated in tumors. Targeted delivery is an important and promising approach for the development of effective therapy in cancer applications. Our main goal is to use 3D whole body tomographic imaging to visualize the receptor-mediated transferrin cellular uptake. Towards this goal, we propose to capitalize on the homodimeric nature of the transferrin receptor and employ Fluorescence Resonance Energy Transfer (FRET) to image in vivo the ability of tissues to take up near-infrared (NIR) iron-bound transferrin. We will use a whole body multispectral time-resolved fluorescence molecular tomography (FMT) imaging platform to measure receptor dimerization in vivo and in 3D. In summary, we will detect and image in vivo receptor-mediated NIR-transferrin cell uptake for the quantitative detection of the transferrin-based targeted delivery systems for diagnostic and therapeutic use. This proposal leapfrogs over current standard approaches to incorporate the most advanced pre-clinical optical imaging approach to assess quantitatively the dimerization of membrane-bound receptors, e,.g. transferrin receptor, by monitoring FRET. FRET FMT imaging works in a non-invasive but in vivo manner, making quantitative data readily available to localize and measure the amount of receptor-mediated transferrin cellular uptake with unprecedented specificity and sensitivity. The long term goal of this research program is to develop a new clinical modality to identify receptor pathways that are activated by receptor dimerization in an individual's cancer. Such non-invasive technique is lacking but will profoundly impact patient management by allowing to devise an individualized therapeutic regimen consisting only of those drugs that will target and block the receptor pathways that are activated in that particular tumor for aggressive and successful therapy. PUBLIC HEALTH RELEVANCE: Transferrin (Tfn) is a well known iron-binding protein, which is responsible for carrying iron into the cells via its binding to the Tfn-receptor (TFR). Importantly, TFR has been shown to be highly expressed in tumors and to promote the growth of breast tumors. Due to the upregulation of TFR in tumors, Tfn has been widely used as carrier for anti-cancer drug/siRNA delivery systems. Targeted delivery is an important and promising approach for the development of effective therapy in cancer applications. Our main goal is to develop a non-invasive whole-body imaging system that can discriminate non-specific receptor-independent tumor accumulation from receptor-mediated uptake into the tumor cells, thus allowing the direct visualization of the delivery of Tfn-targeted particles into tumor cells. Towards this goal, this proposal leapfrogs over current standard approaches by combining fluorescence molecular tomographic (FMT) imaging and Fluorescence Resonance Energy Transfer (FRET) probes to assess quantitatively the receptor-mediated tumor uptake of NIR-Tfn by monitoring in vivo FRET between near-infrared (NIR)-donor-Tfn and NIR-acceptor- Tfn bound to TFR homodimers. Hence, we will image in vivo receptor-mediated NIR-Tfn cell uptake for the quantitative detection of the Tfn-based targeted delivery systems for diagnostic and therapeutic use.